Construction and Identification of Bacterial Artificial Chromosome Library for 0-613-2R in Upland Cotton

A bacterial artificial chromosome （BAC） library containing a large genomlc DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To Isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA Inserts Is needed. We have developed a BAC library of the restoring line 0-613-2R for Isolating the fertility restorer （Rf1） gene and genomic research in cotton （Gossypium hirsutum L.）. The BAC library contains 97 825 clones stored In 255 pieces of a 384-well mlcrotiter plate. Random samples of BACs digested with the Notl enzyme Indicated that the average Insert size Is approximately 130 kb, with a range of 80-275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hlndiii enzymes. Thus, the atabiiity of a single BAC clone can be sustained at iesat for 100 generations. Eight simple sequence repeat （SSR） markers flanking the Rf; gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.     

一种新的脉冲电泳分子量标记*

在脉冲电泳（ｐｕｌｓｅｄ－ｆｉｅｌｄ　ｇｅｌ　ｅｌｃｔｒｏｐｈｏｒｅｓｉｓ,ＰＦＧＥ）研究中,经常使用的分子量标记有啤酒酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｅｓｌａｅ）和粟酒裂殖酵母（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｐｏｍｂｅ）的染色体完整ＤＮＡ。其中,啤酒酵母（如菌株ＹＮＮ２９５）有１６条染色体,分子量变化范围为０．２５Ｍｂ～２．２Ｍｂ（ＭｃＣｌｕｓｋｅｙｅｌａｌ,１９９０）,适于作为小于２．２Ｍｂ的染色体ＤＮＡ的分子量标记;粟酒裂殖酵母（如菌株９７２ｈ－）有３条染色体,分子量…     

The arbuscular mycorrhizal fungus Glomus intraradices is haploid and has a small genome size in the lower limit of eukaryotes

The genome size, complexity, and ploidy of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was determined using flow cytometry, reassociation kinetics, and genomic reconstruction. Nuclei of G. intraradices from in vitro culture, were analyzed by flow cytometry. The estimated average length of DNA per nucleus was 14.07+/-3.52 Mb. Reassociation kinetics on G. intraradices DNA indicated a haploid genome size of approximately 16.54 Mb, comprising 88.36% single copy DNA, 1.59% repetitive DNA, and 10.05% fold-back DNA. To determine ploidy, the DNA content per nucleus measured by flow cytometry was compared with the genome estimate of reassociation kinetics. G. intraradices was found to have a DNA index (DNA per nucleus per haploid genome size) of approximately 0.9, indicating that it is haploid. Genomic DNA of G. intraradices was also analyzed by genomic reconstruction using four genes (Malate synthase, RecA, Rad32, and Hsp88). Because we used flow cytometry and reassociation kinetics to reveal the genome size of G. intraradices and show that it is haploid, then a similar value for genome size should be found when using genomic reconstruction as long as the genes studied are single copy. The average genome size estimate was 15.74+/-1.69 Mb indicating that these four genes are single copy per haploid genome and per nucleus of G. intraradices. Our results show that the genome size of G. intraradices is much smaller than estimates of other AMF and that the unusually high within-spore genetic variation that is seen in this fungus cannot be due to high ploidy.     

Genome size and ploidy level: new insights for elucidating relationships in Zygosaccharomyces species

Ploidy is a fundamental genetic trait with important physiological and genomic implications. We applied complementary molecular tools to highlight differences in genome size and ploidy between Zygosaccharomyces rouxii strain CBS 732T and other related wild strains (ATCC 42981, ABT 301, and ABT 601). The cell cycle analysis by flow cytometry revealed a genome size of 12.7+/-0.2 Mb for strain CBS 732T, 21.9+/-0.2 Mb for ATCC 42981, 28.1+/-1.3 Mb for ABT 301, and 39.00+/-0.3 Mb for ABT 601. Moreover, karyotyping analysis showed a high variability, with wild strains having a higher number of chromosomal bands than CBS 732T. The ploidy level was assessed comparing genome size from flow cytometry with the average haploid size from electrophoretic karyotyping. Strain CBS 732T showed an haploid DNA content, whereas the wild strains a diploid DNA content. In addition gene probe-chromosome hybridization targeted to ZSOD genes showed that wild strains with a diploid DNA content have two ZSOD copies located on different chromosomes.     

Estimation of chromosome number and size by pulsed-field gel electrophoresis (PFGE) in medically important Candida species.

The chromosomal DNAs of eight medically important Candida species, C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata, were analysed by pulsed-field gel electrophoresis under various conditions. The corresponding bands in the gels were assigned by three kinds of DNA probe which hybridized to DNA of all the species: rDNA, TUB2 and PEP4. The best conditions for separating the chromosomal DNAs were investigated and the numbers and molecular sizes of the chromosome bands were determined for each species. The chromosomal DNAs of the species were separated into 5-14 bands ranging in size from 0.5 to 4.5 Mb. Based on the quantification of the chromosome band intensities using a laser fluorescent gel scanner, the chromosome numbers were estimated. The apparent average total number of chromosomes per cell was 16 for C. albicans, 16 for C. stellatoidea, 12 for C. tropicalis, 14 for C. parapsilosis, 8 for C. krusei, 8 for C. guilliermondii, 18 for C.kefyr, and 14 for C. glabrata; the total chromosomal DNA size of each species per cell was calculated at about 31 Mb, 33 Mb, 31 Mb, 26 Mb, 20 Mb, 12 Mb, 29 Mb and 14 Mb, respectively.     

Comparisons with Caenorhabditis (approximately 100 Mb) and Drosophila (approximately 175 Mb) using flow cytometry show genome size in Arabidopsis to be approximately 157 Mb and thus approximately 25% larger than the Arabidopsis genome initiative estimate of approximately 125 Mb

Recent genome sequencing papers have given genome sizes of 180 Mb for Drosophila melanogaster Iso-1 and 125 Mb for Arabidopsis thaliana Columbia. The former agrees with early cytochemical estimates, but numerous cytometric estimates of around 170 Mb imply that a genome size of 125 Mb for arabidopsis is an underestimate. In this study, nuclei of species pairs were compared directly using flow cytometry. Co-run Columbia and Iso-1 female gave a 2C peak for arabidopsis only approx. 15 % below that for drosophila, and 16C endopolyploid Columbia nuclei had approx. 15 % more DNA than 2C chicken nuclei (with >2280 Mb). Caenorhabditis elegans Bristol N2 (genome size approx. 100 Mb) co-run with Columbia or Iso-1 gave a 2C peak for drosophila approx. 75 % above that for 2C C. elegans, and a 2C peak for arabidopsis approx. 57 % above that for C. elegans. This confirms that 1C in drosophila is approx. 175 Mb and, combined with other evidence, leads us to conclude that the genome size of arabidopsis is not approx. 125 Mb, but probably approx. 157 Mb. It is likely that the discrepancy represents extra repeated sequences in unsequenced gaps in heterochromatic regions. Complete sequencing of the arabidopsis genome until no gaps remain at telomeres, nucleolar organizing regions or centromeres is still needed to provide the first precise angiosperm C-value as a benchmark calibration standard for plant genomes, and to ensure that no genes have been missed in arabidopsis, especially in centromeric regions, which are clearly larger than once imagined.     

Unexpected loss of genomic DNA from agarose gel plugs

Intact chromosomal DNAs are routinely prepared by embedding cells in agarose plugs before lysis. The large sizes of the genomic DNAs cause their retention while other macromolecules diffuse into and out of the gel matrix during lysis, washing and restriction cleavage incubations. However, in an analysis of agarose-embedded chromosomal DNAs cleaved with restriction enzymes, fragments larger than 30 kilobases were found to have eluted from the gel plugs. Since loss of fragments from gel plugs may affect qualitative and quantitative interpretations of electrophoretic patterns, an analysis of the diffusion of DNA segments from agarose plugs was performed. The two variables monitored were the time dependence and the DNA fragment size dependence of the diffusion process. The results indicate that small fragments (less than or equal to 2 kilobases) are quickly lost from 1% agarose gel plugs; moreover, significant amounts of large DNA segments (i.e., the 48.5-kilobase lambda phage chromosome) are also lost. In addition to urging caution in the analysis of restriction cleavage data, these observations suggest that intact small organelle genomes and extrachromosomal DNAs also may be lost from genomic DNAs prepared in agarose gel plugs.     

The African trypanosome genome

The haploid nuclear genome of the African trypanosome, Trypanosoma brucei, is about 35 Mb and varies in size among different trypanosome isolates by as much as 25%. The nuclear DNA of this diploid organism is distributed among three size classes of chromosomes: the megabase chromosomes of which there are at least 11 pairs ranging from 1 Mb to more than 6 Mb (numbered I-XI from smallest to largest); several intermediate chromosomes of 200-900 kb and uncertain ploidy; and about 100 linear minichromosomes of 50-150 kb. Size differences of as much as four-fold can occur, both between the two homologues of a megabase chromosome pair in a specific trypanosome isolate and among chromosome pairs in different isolates. The genomic DNA sequences determined to date indicated that about 50% of the genome is coding sequence. The chromosomal telomeres possess TTAGGG repeats and many, if not all, of the telomeres of the megabase and intermediate chromosomes are linked to expression sites for genes encoding variant surface glycoproteins (VSGs). The minichromosomes serve as repositories for VSG genes since some but not all of their telomeres are linked to unexpressed VSG genes. A gene discovery program, based on sequencing the ends of cloned genomic DNA fragments, has generated more than 20 Mb of discontinuous single-pass genomic sequence data during the past year, and the complete sequences of chromosomes I and II (about 1 Mb each) in T. brucei GUTat 10.1 are currently being determined. It is anticipated that the entire genomic sequence of this organism will be known in a few years. Analysis of a test microarray of 400 cDNAs and small random genomic DNA fragments probed with RNAs from two developmental stages of T. brucei demonstrates that the microarray technology can be used to identify batteries of genes differentially expressed during the various life cycle stages of this parasite.     

Electrophoretic karyotyping of the lignin-degrading basidiomycete Phanerochaete chrysosporium

Electrophoretic karyotyping of the two most widely studied strains of Phanerochaete chrysosporium, BKMF-1767 and ME-446, has been determined using transverse alternating field etectrophoresis. The genomic DNA of BKMF-1767 was resolved into 10 chromosomes ranging in size from 1.8–5.0 Mb, amounting to a total genome size of about 29 Mb. The genomic DNA of strain ME-446, on the other hand, was resolved into 11 chromosomes, amounting to a total genome size of about 32Mb. Lignin peroxidase genes have been localized to five chromosomes in strain BKMF-1767 and to four chromosomes in strain ME-446.     

Electrophoretic karyotypes of Phaffia rhodozyma strains

Abstract Electrophoretic karyotypes of strains from the astaxanthin-producing yeast Phaffia rhodozyma have been established. Intact chromosomal DNA molecules released from protoplasts were separated by orthogonal field alternation gel electrophoresis (OFAGE) and contour clamped homogeneous electric field (CHEF). Both small and large chromosomal DNA molecules were resolved simultaneously by optimizing the running conditions. Electrophoretic karyotypes among the Phaffia isolates examined differed significantly. Seven to thirteen chromosomal bands, ranging in size from 0.83 Mb to 3.50 Mb, were resolved, giving total genome sizes of about 15.4 to 23.2 Mb. Ribosomal DNA has been assigned to chromosomal bands using a heterologous gene probe.     

Electrophoretic karyotype of Saprolegnia monoica

Abstract The electrophoretic karyotype of Saprolegnia monoica was determined by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Eight chromosomal bands were separated. The size of these bands, based on migration relative to those of chromosomal DNA of Saccharomyces cerevisiae , Schizosaccharomyces pombe and Hansenula wingei , is estimated to be between 0.9 and 5.8 Mb. The genome size is estimated to be 51 Mb.     

Evidence for a chromosomal location of polydnavirus DNA in the ichneumonid parasitoid Hyposoter fugitivus.

Evidence is presented in support of a chromosomal location for sequences homologous to polydnavirus DNA in the ichneumonid parasitoid Hyposoter fugitivus. In this study, four different viral genome segments were cloned and used as probes against genomic DNA extracted from male parasitoids and digested with a variety of restriction enzymes. Each probe typically identified a single off-size fragment (OSF) in the case of enzymes not cutting viral genome segments, while two OSFs were generated by enzymes cutting at one and two sites. While extra OSFs were occasionally observed, these were invariably found to be due to the presence of polymorphic restriction sites in flanking chromosomal DNA. Analysis of these data suggests that a single, stable chromosomal locus exists for sequences homologous to each viral genome segment; the data also indicate that viral and cognate parasitoid genomic DNAs are largely if not entirely colinear.     

镰孢属几种植物病原真菌分子核型的研究*

利用脉冲电场凝胶电泳（ｐｕｌｓｅｄ－ｆｉｅｌｄｇｅｌｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ,ＰＦＧＥ）,研究了４株串珠镰孢（Ｆｕｓａｒｉｕｍ　ｍｏｎｉｌｉｆｏｒｍｅ）、１株尖镰孢（F．ｏｘｙｓｐｏｒｕｍ）、１株茄镰孢（Ｆ．ｓｏｌａｎｉ）和１株Ｆｕｓａｒｉｕｍｓｐ．的分子核型以及不同地域和寄主来源的串珠镰孢种内菌株间的分子核型差异。以凝胶包埋法（不破除分生孢子细胞壁）制备供试菌株电泳样本,采用３组条件组合进行电泳,分离出供试串珠镰孢完整染色体ＤＮＡ１０～１３条,分子量分布范围０．７Ｍｂ～６．９Ｍｂ,基因组大小为４２．２６Ｍｂ～４７．７５Ｍｂ;尖镰孢８条,分子量分布范围１．２Ｍｂ～６．７Ｍｂ,基因组大小为３２．２５Ｍｂ;茄镰孢６条,分子量分布范围２．４Ｍｂ～６．３Ｍｂ,基因组大小为２５．２Ｍｂ;Ｆｕｓａｒｉｕｍｓｐ．９条,分子量分布范围０．８Ｍｂ～６．８Ｍｂ,基因组大小为３６．４５Ｍｂ。结果表明,供试４种镰孢菌染色体数目、ＤＮＡ分子量及基因组大小都有较大不同,分子核型差异较大。不同来源的串珠镰孢种内菌株间分子核型亦有明显差异。     

Separation of megabased-sized DNA molecules of Aspergillus niger using pulsed field gel electrophoresis

In this study, the chromosomal DNAs were extracted from Aspergillus niger Z10 wild type strain and these DNAs were separated using the contour clamped homogeneous electric field gel electrophoresis (CHEF) system. This system is laboratory-made and is operated by a computer program. Total DNAs resolved into five distinct chromosomal bands. The size of the chromosomes was estimated as being between 3.3 Mb to 6.4 Mb.     

镰孢属几种植物病原真菌分子核型的研究

利用脉冲电场凝胶电泳（ｐｕｌｓｅｄ－ｆｉｅｌｄｇｅｌｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ,ＰＦＧＥ）,研究了４株串珠镰孢（Ｆｕｓａｒｉｕｍ　ｍｏｎｉｌｉｆｏｒｍｅ）、１株尖镰孢（F．ｏｘｙｓｐｏｒｕｍ）、１株茄镰孢（Ｆ．ｓｏｌａｎｉ）和１株Ｆｕｓａｒｉｕｍｓｐ．的分子核型以及不同地域和寄主来源的串珠镰孢种内菌株间的分子核型差异。以凝胶包埋法（不破除分生孢子细胞壁）制备供试菌株电泳样本,采用３组条件组合进行电泳,分离出供试串珠镰孢完整染色体ＤＮＡ１０～１３条,分子量分布范围０．７Ｍｂ～６．９Ｍｂ,基因组大小为４２．２６Ｍｂ～４７．７５Ｍｂ;尖镰孢８条,分子量分布范围１．２Ｍｂ～６．７Ｍｂ,基因组大小为３２．２５Ｍｂ;茄镰孢６条,分子量分布范围２．４Ｍｂ～６．３Ｍｂ,基因组大小为２５．２Ｍｂ;Ｆｕｓａｒｉｕｍｓｐ．９条,分子量分布范围０．８Ｍｂ～６．８Ｍｂ,基因组大小为３６．４５Ｍｂ。结果表明,供试４种镰孢菌染色体数目、ＤＮＡ分子量及基因组大小都有较大不同,分子核型差异较大。不同来源的串珠镰孢种内菌株间分子核型亦有明显差异。     

Characterisation of DNA forms associated with cassava latent virus infection.

In addition to the major encapsidated DNA species found in preparations of cassava latent virus (genomic DNAs 1 and 2) there are minor DNA populations of twice (dimeric) and approximately half genome length. Both minor species resemble the genomic DNAs in that they are composed of predominantly circular single-stranded DNA. All of these size groups have a corresponding covalently-closed circular double-stranded DNA form in infected tissue. Infectivity studies using cloned DNAs 1 and 2 show that dimeric DNA routinely appears, suggesting it to be an intermediate in the DNA replicative cycle that can be encapsidated at low efficiency. In contrast, half unit length DNA has not yet been detected after multiple passaging of virus derived from the cloned DNA inoculum. Half unit length DNAs appear to be derived exclusively from DNA 2 and consist of a population of molecules exhibiting a relatively specific deletion. As they have an inhibitory effect on virus multiplication, their encapsidated forms are analogous to defective interfering particles associated with other eukaryotic DNA containing viruses. Small primer molecules associated with the genomic single-stranded DNAs, as reported for another geminivirus, have not been detected in CLV.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Incongruent evolution of chromosomal size in rice

To investigate genome size evolution, it is usually informative to compare closely related species that vary dramatically in genome size. A whole genome duplication (polyploidy) that occurred in rice (Oryza sativa) about 70 million years ago has been well documented based on current genome sequencing. The presence of three distinct duplicate blocks from the polyploidy, of which one duplicated segment in a block is intact (no sequencing gap) and less than half the length of its syntenic duplicate segment, provided an excellent opportunity for elucidating the causes of their size variation during the post-polyploid time. The results indicated that incongruent patterns (shrunken, balanced and inflated) of chromosomal size evolution occurred in the three duplicate blocks, spanning over 30 Mb among chromosomes 2, 3, 6, 7, and 10, with an average of 20.3% for each. DNA sequences of chromosomes 2 and 3 appeared to had become as short as about half of their initial sequence lengths, chromosomes 6 and 7 had remained basically balanced, and chromosome 10 had become dramatically enlarged (approximately 70%). The size difference between duplicate segments of rice was mainly caused by variations in non-repetitive DNA loss. Amplification of long terminal repeat retrotransposons also played an important role. Moreover, a relationship seems to exist between the chromosomal size differences and the nonhomologous combination in corresponding regions in the rice genome. These findings help shed light on the evolutionary mechanism of genomic sequence variation after polyploidy and genome size evolution.     

Size and complexity of the nuclear genome of the ectomycorrhizal fungus Paxillus involutus

The basidiomycete Paxillus involutus is forming ectomycorrhizal symbiosis with a broad range of forest trees. Reassociation kinetics on P. involutus nuclear DNA indicated a haploid genome size of 23 Mb including 11% of repetitive DNA. A similar genome size (20 Mb) was estimated by genomic reconstruction analysis using three single copy genes. To assess the gene density in the P. involutus genome, a cosmid containing a 33-kb fragment of genomic DNA was sequenced and used to identify putative open reading frames (ORFs). Twelve potential ORFs were predicted, eight displayed significant sequence similarities to known proteins found in other organisms and notably, several homologues to the Podospora anserina vegetative incompatibility protein (HetE1) were found. By extrapolation, we estimate the total number of genes in the P. involutus haploid genome to approximately 7700.     

Genomic methylation: a tool for typing Helicobacter pylori isolates

The genome sequences of three Helicobacter pylori strains revealed an abundant number of putative restriction and modification (R-M) systems within a small genome (1.60 to 1.67 Mb). Each R-M system includes an endonuclease that cleaves a specific DNA sequence and a DNA methyltransferase that methylates either adenosine or cytosine within the same DNA sequence. These are believed to be a defense mechanism, protecting bacteria from foreign DNA. They have been classified as selfish genetic elements; in some instances it has been shown that they are not easily lost from their host cell. Possibly because of this phenomenon, the H. pylori genome is very rich in R-M systems, with considerable variation in potential recognition sequences. For this reason the protective aspect of the methyltransferase gene has been proposed as a tool for typing H. pylori isolates. We studied the expression of H. pylori methyltransferases by digesting the genomic DNAs of 50 strains with 31 restriction endonucleases. We conclude that methyltransferase diversity is sufficiently high to enable the use of the genomic methylation status as a typing tool. The stability of methyltransferase expression was assessed by comparing the methylation status of genomic DNAs from strains that were isolated either from the same patient at different times or from different stomach locations (antrum and corpus). We found a group of five methyltransferases common to all tested strains. These five may be characteristic of the genetic pool analyzed, and their biological role may be important in the host/bacterium interaction.